This invention claims priority of a German patent application DE-199 56 438.8 which is incorporated by reference herein.
The present invention concerns an arrangement for scanning a specimen receiving device for data recording with a laser scanning microscope, preferably with a confocal laser scanning microscope.
In laser scanning microscopes, exciting light from a laser is focused onto a specimen and the intensity of the detected light from that focus position is detected with a detector. In order to obtain a two- or three-dimensional image of the specimen, either the focused laser beam is scanned over the specimen (beam scanning) or the specimen is moved through the focus position (specimen scanning). Beam scanning is usually implemented with a scanning mirror arranged movably in the beam path of a laser scanning microscope; this allows relatively rapid data recording. For certain applications, however, the maximum specimen field that can be recordedxe2x80x94which is defined by the microscope optical system usedxe2x80x94is too small. No limitation in terms of specimen field exists with specimen scanning, in which a specimen holder is moved in a meander pattern through the focus position; this is generally implemented with mechanically complex X-Y displaceable stages. Large masses must be accelerated in this context, however, with the result that data recording is time-intensive and is associated with a high outlay in terms of control technology. Data recording is considerably slower with specimen scanning, and this is unacceptable for routine applications.
It is therefore the object of the present invention to provide an arrangement, with which imaging and scanning of large specimen fields can be performed at sufficient speed; this is also, in particular, to be possible with the use of simple microscope optics.
The aforesaid object is achieved by an arrangement for scanning a specimen comprising:
a specimen receiving device for data recording
a laser scanning microscope,
a rotation device, defining a first axis, for alternatingly rotating the specimen receiving device about the first axis and
a second axis is defined in the rotation device for rotating the specimen receiving device about the second axis.
According to the present invention, what has been recognized first of all is that imaging of large specimen fields can be achieved with specimen scanning, in particular using simple microscope optics, if the imaging speed hitherto attainable can be increased. According to the present invention, for this purpose the specimen receiving device is not moved in a linear direction through the focus, but rather is alternatingly rotated about a first axis by a rotation device.
The term xe2x80x9calternatingly rotatablexe2x80x9d is to be understood in this connection to mean that the specimen receiving device is first rotated clockwise about a rotation axis, and then counter-clockwise about the same rotation axis. In other words, the resulting rotary movement is made up of recurring individual rotations in opposite directions. The rotation of the specimen receiving device is effected by a suitable rotation device. With a rotation of this kind, a recurring data recording of a corresponding xe2x80x9cone-dimensionalxe2x80x9d circular segment of the specimen is detected.
A data recording going beyond one circular segment is made possible, in an advantageous embodiment, by the fact that the specimen receiving device is rotated, together with the rotation device, about a second axis. This makes possible two- or three-dimensional data recording from the specimen. In physical terms, the specimen receiving device could be attached, together with the rotation device of the first axis, to a retainer that is mounted rotatably about the second axis. If the first rotation axis is arranged at least almost parallel to the second rotation axis, it is thereby possible to achieve a scanning motion of the specimen that lies in one plane.
Advantageously, the two rotation axes are arranged relative to the optical axis of the laser scanning microscope in such a way that the resulting scan trajectories of the two axes extend almost orthogonally to one another. The term xe2x80x9cscan trajectoryxe2x80x9d is to be understood in this context as the line pattern, projected by the laser scanning microscope onto the specimen, that results from the scanning motion of the specimen receiving device. It represents the coordinate system of the image data after digitization. If the two rotation axes are arranged such that their scan trajectories each extend almost orthogonally to one another, the result is optical scanning of the specimen at a scanning rate (and a resolution and therefore information density) that is almost spatially uniform. This is important above all in terms of subsequent processing of the recorded image data, since the latter are present, after a data recording, in the curvilinear coordinate system and, for example after a coordinate transformation into a rectilinear coordinate system, also possess a largely uniform information density.
Advantageously, it is possible to move the specimen receiving device, together with the rotation device, in translational fashion so as thereby to produce a two- or three-dimensional data recording or scan of a specimen.
In a concrete embodiment, the resulting scanning motion of the specimen receiving device lies in one plane. Two-dimensional regions of three-dimensional specimens can thus be imaged or scanned.
In a further embodiment, the scanning motion of the specimen receiving device extends at least almost parallel to the surface of the specimen receiving device. If the specimen to be detected is located directly beneath the surface of the specimen receiving device, by way of this feature it is possible for the specimen to be completely imaged by scanning a single plane, provided the specimen thickness and the depth of the field of the microscope optics are of the same order of magnitude.
If the specimen receiving device is moved translationally together with the rotation device, in an alternative embodiment provision is made for the translational movement to extend at least almost parallel to the surface of the specimen receiving device. This can again produce a scanning motion which then lies in one plane and extends parallel to the surface of the specimen receiving device. This, too, would advantageously make possible a complete data recording of a specimen located directly beneath the surface of the specimen receiving device.
If it is necessary to image three-dimensional specimens whose extension along the optical axis is greater than the depth of field of the microscope optics, provision is made for a translational motion of the specimen receiving device along the optical axis. It is thus ultimately possible, by way of the combination of rotational and translational motions, to use specimen scanning to record a complete image of a three-dimensionally extending specimen.
In a concrete embodiment, the translational motion extends along one linear direction. In particular, the translational motion could extend perpendicular to the first rotation axis. Concretely, the translational motion extends periodically in opposite directions, i.e. what is present is a recurring back-and-forth movement of the specimen receiving device together with the rotation device. This could be implemented, for example, by way of a linear displacement stage having corresponding guidance means.
In particularly advantageous fashion, the specimen receiving device is arranged with respect to the optical axis of the laser scanning microscope in such a way that the line normal to the surface of the specimen receiving device forms an angle with the optical axis of the laser scanning microscope that differs from 0 degrees. The principal return reflection of the exciting light, which occurs for example at the optical transition to the specimen receiving device, can thus advantageously be suppressed or blocked out from the excitation or detection beam path of the laser scanning microscope. This is important in particular because a (bandpass) blocking filter of lesser strength can now be used, which only insignificantly reduces the fluorescent light being detected. Blocking out the principal return reflection is advantageous in particular when lasers are used, since the exciting light returning to the laser generally disrupts its stimulated emission, which can result in undesirable intensity fluctuations in the laser light. In addition, it is possible to prevent reflections from specimen holder edges that can result in disruptive interference in the specimen region and thus also cause imaging artifacts.
The reflected component of the exciting light that is blocked out in this fashion therefore does not arrive at the detector, thus making possible an increase in the dynamic range of the detected signal. Corresponding blocking filters that are arranged in front of the detector in order to filter scattered or reflected components of the exciting light out of the detection beam path can thus advantageously be selected in such a way that the detected light is attenuated less by these filters placed in front of the detector. Advantageously, the angle between the line normal to the surface of the specimen receiving device and the optical axis of the laser scanning microscope is greater than 0 and less than 10 degrees.
The specimen receiving device has a specimen holder and a specimen holder unit. The specimen holder receives the specimen being detected and can be, for example, a conventional specimen holder made of glass. The specimen on the glass specimen holder could be covered with a cover slip. In very general terms, the specimen holder can have a glass plate on which the specimens to be imaged are arranged or wax-mounted. This glass plate is then integrated into a corresponding chamber unit.
The specimen receiving device could be automatically loaded with specimen holders, thus making possible, in automated laboratory use, a high throughput of specimen holders for examination.
The specimen holder unit and the specimen holder are produced from material with a low density/weight, preferably from plastic or aluminum. Lightweight design for these constituents allows a high scanning frequency for the rotation device, so that the data recording time can advantageously be reduced.
The specimen receiving device that is to be scanned is configured such that its center of gravity lies on the first or second rotation axis. With a physical configuration of this kind, no additional torques occur during scanning, the principal result thereof being that minimization of the vibrations of the specimen receiving device brought about by the scanning operation can be achieved.
In terms of automatic loading of the specimen receiving device with specimen holders, the specimen holder could be attached to the specimen holder unit with at least one clamping apparatus. A conventional metal clamping spring which presses the specimen holder onto a support of the specimen holder unit could be used, for example, as the clamping apparatus.
The specimen holder could also be attached to the specimen holder unit with the aid of a negative-pressure or vacuum device. The negative-pressure or vacuum device could, for example, be arranged in stationary fashion on the laser scanning microscope and connected via a flexible hose to the specimen holder unit in order to act upon the specimen holder with negative pressure.
It would furthermore be conceivable for the specimen holder to be attachable to the specimen holder unit on the basis of magnetic or electromagnetic interaction. For that purpose, for example, a portion of the specimen holder could be made of ferromagnetic material that can be attached to a metallic or also ferromagnetic specimen holder unit. It is also conceivable for the specimen holder unit to be configured electromagnetically so that a specimen holder made at least partially of metal can be attached by electromagnetic interaction to the specimen holder unit.
Advantageously, a galvanometer is used as the rotation device of the specimen receiving device. This is advantageous in particular because galvanometers, as compared to linear displacement stages, are economically, easy to control, and commercially available in a wide variety of specifications. The galvanometer that rotates the specimen receiving device can accordingly be selected and dimensioned so that the highest possible oscillation frequency is achievable with minimum vibration phenomena in the specimen scanning system. In this context, any deviation from the reference or actual position of the galvanometer, or deviation in scanning speed, should be minimal. The galvanometer is operated at a frequency in the range from 10 to 1000 Hz. If the rotation device is to be operated at a constant scanning frequency, a resonant galvanometer could be used. This would offer the advantage that the oscillation produced by the resonant galvanometer corresponds practically exactly to the reference frequency of the resonant galvanometer.
A galvanometer could also be provided as the further rotation device for the second axis. This galvanometer is dimensioned in such a way that it rotates the specimen receiving device together with the rotation device of the first axis. This galvanometer is accordingly operated at a lower scanning frequency. Ideally, the physical arrangement of the assemblies to be rotated by this further galvanometer is selected in such a way that, in order to avoid additional torques, their center of gravity is located on the axis of the further galvanometer.
In a further embodiment, a lever device is provided as the rotation device for the second axis. The lever device has an electric motor, a threaded spindle, a recirculating threaded piece, and a connecting element. The threaded spindle is associated with the shaft of the electric motor, and a circulating threaded coupling located on the threaded spindle can be positioned, by rotation of the threaded spindle, along the direction of the axis of the threaded spindle; no inherent rotation is provided for the circulating threaded coupling. The circulating threaded coupling is connected to the rotation device via a connecting element; the connecting element could, for example, be embodied as a leaf spring. Advantageously, torque transfer of the linear motions of the circulating threaded coupling to the rotation device by way of the leaf spring is accomplished with zero play, with the result that a well-defined and, above all, reproducible rotary motion can be performed.
If the electric motor is arranged in stationary fashion, rotation of the shaft and of the threaded spindle moves the circulating threaded coupling in the direction of the axis of the threaded spindle; as a result, with reference to the second rotation axis of the specimen receiving device, a torque is transferred to the specimen receiving device, so that the latter is thereby rotated about the second axis. Advantageously, the electric motor is attached in such a way that it is mounted in resiliently pivotable fashion about an axis. This axis could be arranged perpendicular to the rotation axis of the electric motor. Any slight imbalance in the shaft of the electric motor or any slight mechanical disruption of the specimen scanning system can be compensated for by the resilient mounting of the electric motor, so that these disruptions are not transferred to the rotation device, for example in the form of undesirable motions.
Advantageously, the arrangement for scanning the specimen receiving device is vibrationally decoupled from the laser scanning microscope. The principal consequence of this is to prevent any misalignment of individual optical components of the laser scanning microscope that might be brought about due to vibrations of the rotation device.
Data recording of specimens could be performed unidirectionally in terms of the rapid rotation axis. For example, in the case of an alternating rotation of the specimen receiving device, a data recording could be accomplished only when the specimen receiving device is, for example, rotating clockwise, but not when the specimen receiving device is rotating counter-clockwise.
Data recording could also be accomplished bidirectionally in terms of the rapid rotation axis. Image data are thus recorded during both clockwise and counter-clockwise rotation. No provision is made here for data recording at the locations of the reversing points, i.e. the selected rotation region of the specimen receiving device must be larger than the specimen region that is to be imaged.
The image data recorded with the aid of the rotation device are present, after digitization with a digitization device in the control computer of the laser scanning microscope, in a curvilinear coordinate system. These image data are transformed with a computer program module into a rectilinear coordinate system. If the specimen receiving device is rotated, for data recording, in each case about a first and a second axis, the transformation can be performed according to the equation       (                            x                                      y                      )    =                    r        1            ⁡              (                                                            sin                ⁢                                  xe2x80x83                                ⁢                α                                                                                                          cos                  ⁢                                      xe2x80x83                                    ⁢                  α                                -                1                                                    )              +                            r          2                ⁡                  (                                                                      cos                  ⁢                                      xe2x80x83                                    ⁢                  α                                                                              sin                  ⁢                                      xe2x80x83                                    ⁢                  α                                                                                                                          -                    sin                                    ⁢                                      xe2x80x83                                    ⁢                  α                                                                              cos                  ⁢                                      xe2x80x83                                    ⁢                  α                                                              )                    ⁢              xe2x80x83            ⁢              (                                                                              cos                  ⁢                                      xe2x80x83                                    ⁢                  β                                -                1                                                                                        sin                ⁢                                  xe2x80x83                                ⁢                β                                                    )            
where xcex1 and xcex2 are the angular positions of the respective rotation axes; r1, r2 are the radii of the respective rotation axes; and x, y are the coordinates of an image point after transformation.
In terms of a concrete embodiment, the transformation is performed at least partially with the aid of an input lookup table of the digitization device. For example, the function values of the functions sin xcex1 and sin xcex2 could be stored in the input lookup table.
In addition, the corresponding matrix multiplication of the transformation equation could be performed in a field-programmable gate array (FPGA) module, downstream from the input lookup table, in which the constants r1 and r2 are also stored. This makes possible, in particular, transformation simultaneously with data recording, which in turn allows high data throughput.
If provision is made for the use of immersion oil for microscopic imaging, the oil is advantageously pipetted (preferably automatically), onto the specimen holder before the automatic loading operation. The immersion oil could, for example, be water, glycerol, or conventional immersion oil, but depending on its viscosity an upper limit is then placed on the maximum scanning speed of the specimen receiving device.
In a concrete embodiment, the laser scanning microscope has a stationary illumination and detection beam. As a result, complex field correction of a microscope objective that is used is not necessary, and a relatively simple and economical objective can be used. This procedure is advantageous especially in terms of alignment of the optical components, since the laser scanning microscope needs to be aligned only during production, and requires no further alignment thereafter.
For allocation of the scanned image data to the coordinates of the respective scan position, the rotation device has a position transducer whose output is connected to the control unit of the laser scanning microscope. This position transducer supplies the instantaneous angular positions of the rotation axes necessary for the transformation equation, so that a detected image point can be allocated with high accuracy to its actual position coordinate. If a translational motion is provided for scanning of the specimen receiving device, the device for performing the translational motion analogously has a position transducer whose output is also connected to the control unit of the laser scanning microscope.